The processivity of a polymerase, i.e., the amount of product generated by the enzyme per binding event, can be enhanced by increasing the stability of the modifying enzyme/nucleic acid complex. The current invention now provides enhanced polymerase assays that employ novel modifying enzymes in which the double-stranded conformation of the nucleic acid is stabilized and the processivity of the enzyme increased by joining a sequence-non-specific double-stranded nucleic acid binding domain to the enzyme, or its catalytic domain which are disclosed e.g., in co-pending U.S. application Ser. No. 09/870,353 and WO01/92501. The modifying proteins that are processive in nature exhibit increased processivity when joined to a binding domain compared to the enzyme alone.
There is a need to enhance polymerase reactions in many applications. For example, SYBR Green I (Molecular Probes, Eugene, Oreg.; U.S. Pat. Nos. 5,436,134 and 5,658,751), a fluorescent dye that is specific for dsDNA detection, is widely used in real-time PCR reactions to monitor the generation of dsDNA through each cycle of amplification. However, the addition of SYBR Green I inhibits the activity of DNA polymerases used in PCR. Similarly, it is often desirable to use PCR for the analysis of crude or “dirty” nucleic acid samples. For example, colony PCR is a useful technique in which small samples of single bacterial colonies are lysed and added directly to PCR reactions for the purpose of screening colonies for particular DNA sequences. However, colony PCR has a high failure rate, because of residual contaminants from the colony. Thus, polymerases that are resistant to such inhibitors, e.g., fluorescent dyes and impurities present in the cell extracts, are needed in order to obtain more efficient polymerase reactions, e.g., PCR.
There is also a need to improve sequencing reactions. Polymerases currently employed in sequencing reactions, e.g., cycle sequencing, are often inefficient. For example, cycle sequencing is often performed with poorly-processive enzymes. Often, the enzymes used are ΔTaq derivatives, which have Taq polymerase's 5′-3′ nuclease domain removed, and have a processivity of about 2 bases. Also, in the case of dye terminator-sequencing, dITP is used in place of dGTP, which causes polymerase pausing and dissociation at G nucleotides. These enzymes therefore produce a large number of sequence products that are improperly terminated. These stops compete with, and negatively effect, the production of properly terminated sequence products. Furthermore, if a polymerase dissociates during primer extension of a template containing a repeat unit (e.g., a triplet repeat) or secondary structure (e.g., a stem and loop), the 3′ end can denature and reanneal so as to prime at a different location on the template—for example, in the case of a repeat, the reannealing could occur at a different repeat; or in the case of secondary structure, improper reannealing could delete out a section of the template. Thus, dissociation of the polymerase during sequencing can cause a problem in efficiently obtaining reliable sequencing information.
The current invention addresses both of these needs, i.e., the need for enhancing polymerase reactions performed in the presence of inhibitors and the need for enhancing processivity in DNA sequencing applications). The current invention provides such enhanced, or improved, polymerase reactions. The improvement is the use of a polymerase that has increased processivity due to the presence of a sequence-non-specific nucleic-acid-binding domain that is joined to the polymerase.